Alkaline protease

ABSTRACT

An alkaline protease having an amino acid with one or more amino acid residues selected from among those located at (a) position 15, (b) position 16, (c) position 166, (d) position 167, (e) position 187, (f) position 346, and (g) position 405 of the amino acid sequence of SEQ No; 1, or at positions corresponding to these positions, are the following amino acid residues, respectively: (a): histidine, (b): threonine or glutamine, (c): glycine, (d): valine, (e): serine, (f): arginine, and (g); and a gene encoding the alkaline protease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit to priority under 35 U.S.C.§119(a)-(d) to JP 2003-106709, filed Apr. 10, 2003.

FIELD OF THE INVENTION

The present invention relates to an alkaline protease and to a geneencoding the same.

BACKGROUND OF THE INVENTION

Protease has long been used in industry, and has found utility in adiversity of fields, including detergents such as laundry detergents,fiber modifying agents, leather processing agents, cosmeticcompositions, bath additives, food-modifying agents, andpharmaceuticals. Of these, proteases for detergent use are produced inthe largest amounts on an industrial scale. Examples of such knownproteases that are derived from Bacillus include ALCALASE, SAVINASE(registered trademarks; Novozymes), MAXACAL (registered trademark;Genencor), BLAP (registered trademark; Henkel), and KAP (KaoCorporation).

The purpose of incorporating protease into a detergent is to degradeprotein soil adhering to clothes. Such soil actually is a “complex” soilformed of a plurality of organic and inorganic components, including notonly proteins but also lipids originating from sebum, solid particles,and other substances. Therefore, demand has arisen for a detergenthaving excellent detergency to such complex soil.

Under the above situation, some of the present inventors had previouslydiscovered several species of alkaline protease which have a molecularweight of about 43,000, exhibit a sufficient casein-degrading activityeven in the presence of a fatty acid at a high concentration, and alsoexhibit excellent detergency not only to proteins but also to complexsoils which include sebum and other substances, and filed a patentapplication therefor (see Patent Publication WO99/18218). Since thediscovered alkaline proteases differ from subtilisin (which is aconventionally known serine protease derived from a microorganismbelonging to the genus Bacillus) in terms of molecular weight, primarystructure, enzymological characteristics and resistance to oxidants (thealkaline proteases are strongly resistant to oxidants) theirclassification into a new subtilisin subfamily has been proposed (see,for example, Saeki et al., Biochem. Biophys. Res. Commun., 279, 313-319,2000).

In order to industrially mass-produce protease having excellentdetergency, productivity thereof must be enhanced. To this end, avariety of methods are envisaged, including selective mutant breeding ofenzyme-producing bacteria, and alteration of a gene coding for proteaseor a gene related to regulation of expression thereof so as to increasethe amount of secreted protein. Alternatively, the gene coding forprotease is modified so that an enhanced specific activity is obtained.From these viewpoints, the present inventors have previously discoveredmutated alkaline protease exhibiting improved protein secretion abilityand enhanced specific activity (Japanese Patent Application Nos.14-304230, 14-304231, and 14-304232 Application Laid-Open (kokai) Nos.2004-000122 and 2004-057195).

However, further improvement in productivity is desired for producingthe enzyme on a large scale at low cost. To answer this, means forimproving the amount of secreted protein and specific activity hasbecome of keen interest.

SUMMARY OF THE INVENTION

The present invention provides an alkaline protease having an amino acidsequence wherein one or more amino acid residues selected from thoselocated at (a) position 15, (b) position 16, (c) position 166, (d)position 167, (e) position 187, (f) position 346, and (g) position 405of the amino acid sequence of SEQ ID NO: 1, or at positionscorresponding to these positions are the following amino acid residues,respectively:

-   -   Position (a): histidine,    -   Position (b): threonine or glutamine,    -   Position (c): glycine,    -   Position (d): valine,    -   Position (e): serine,    -   Position (f): arginine, and    -   Position (g): aspartic acid.

The present invention also provides a gene encoding the alkalineprotease.

The present invention also provides a vector comprising the gene, and atransformant containing the vector.

The present invention also provides a detergent composition containingthe above-described alkaline protease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-1 to FIG. 1-5 shows amino acid sequence alignment of proteasehaving 80% or higher homology with the amino acid sequence of SEQ ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an alkaline protease which has forexample excellent detergency against complex soil, exhibits enhancedprotein secretion amount and enhanced specific activity, and can beproduced at high productivity.

The present inventors have searched for a new enzyme which is endowedwith the characteristics of the aforementioned alkaline protease andalso with improved protein secretion amount and specific activity, andhave found that such an enzyme, which is a certain alkaline protease,requires the presence of specified amino acid residue(s) at specifiedposition(s) of the amino acid sequence of the alkaline protease.

The alkaline protease of the present invention has an amino acidsequence wherein one or more amino acid residues selected from thoselocated at (a) position 15, (b) position 16, (c) position 166, (d)position 167, (e) position 187, (f) position 346, and (g) position 405of the amino acid sequence of SEQ ID NO: 1, or at positionscorresponding to these positions are the following amino acid residues,respectively:

-   (a): histidine, (b): threonine or glutamine, (c): glycine, (d):    valine, (e): serine, (f): arginine, and (g): aspartic acid.

Namely, the alkaline protease of the present invention is a proteasethat has been engineered such that one or more amino acid residuesselected from among the above-mentioned positions (a) to (g) of analkaline protease having an amino acid sequence of SEQ ID NO: 1, oramino acid residue(s) of another alkaline protease at position(s)corresponding to the above-mentioned positions (a) to (g), are specifiedamino acid residue(s), and may be of a wild type, mutant(s) of the wildtype, or mutant(s) created by artificial mutagenesis.

As used herein, “another alkaline protease” may be either a wild typeenzyme or a mutant of the wild type enzyme. Preferably, “anotheralkaline protease” exhibits resistance to oxidants and has a molecularweight of 43,000±2,000 as determined by SDS-PAGE (sodium dodecylsulfatepolyacrylamide gel electrophoresis), and as an example thereof, mentionmay be given of an alkaline protease having such an amino acid sequencethat exhibits 80% or higher homology with the amino acid sequence of SEQID NO: 1. More preferably, “another alkaline protease” is an enzymewhich has an amino acid sequence that exhibits 80% or higher homologywith the amino acid sequence of SEQ ID NO: 1; acts in an alkaline regionof pH 8 or higher; exhibits resistance to oxidants; shows 80% or higherresidual activity after treatment at 50° C. for 10 minutes at pH 10; isinhibited by diisopropylfluorophosphate (DFP) and phenylmethanesulfonylfluoride (PMSF); and has a molecular weight of 43,000±2,000 asdetermined by SDS-PAGE. As used herein, the expression “exhibitresistance to oxidants” means that after alkaline protease is left tostand at 30° C. for 20 minutes in 20 mM Britton-Robinson buffer (pH 10)containing 50 mM hydrogen peroxide and 5 mM calcium chloride, thealkaline protease maintains a residual activity of at least 50%.

Examples of the “alkaline protease having an amino acid sequence of SEQID NO: 1” include KP43 (derived from Bacillus sp. KSM-KP43 (FERMBP-6532), Patent Publication WO99/18218) (SEQ ID NOS: 12 and 13).Examples of the “alkaline protease having an amino acid sequence thatexhibits 80% or higher homology with the amino acid sequence of SEQ IDNO: 1” include protease KP9860 (GenBank Accession No. AB046403) (derivedfrom Bacillus sp. KSM-kp9860 (FERM BP-6534), Patent PublicationWO99/18218); protease 9865 (GenBank Accession No. AB084155) (derivedfrom Bacillus sp. KSM-9865 (FERM P-1592), Japanese Patent ApplicationLaid-Open (kokai) No. 2003-199559) (SEQ ID NOS: 14 and 15; protease E-1(GenBank Accession No. AB046402) (derived from Bacillus No. D-6 (FERMP-1592), Japanese Patent Application Laid-Open (kokai) No. 49-71191)(SEQ ID NOS: 16 and 17); protease Ya (GenBank Accession No. AB046404)(derived from Bacillus sp. Y (FERM BP-1029), Japanese Patent ApplicationLaid-Open (kokai) No. 61-280268) (SEQ ID NOS: 18 and 19); protease SD521(GenBank Accession No. AB046405) (derived from Bacillus SD521 (FERMP-11162), Japanese Patent Application Laid-Open (kokai) No. 3-191781)SEQ ID NOS: 20 and 21; protease A-1 (GenBank Accession No. AB046406)(derived from NCIB12289, Patent Publication WO88/01293) (SEQ ID NOS: 22and 23); protease A-2 (derived from NCIB12513, Patent PublicationWO98/56927) (SEQ ID NOS: 24 and 25); mutant proteases described inJapanese Patent Application Laid-Open (kokai) Nos. 2002-218989 and2002-306176; mutants obtained through substitution of position 251 ofthe amino acid sequence of SEQ ID NO: 1 by asparagine, threonine,isoleucine, valine, leucine or glutamine; mutants obtained throughsubstitution of position 256 of the same amino acid sequence by serine,glutamine, asparagine, valine, or alanine (Japanese Patent ApplicationLaid-Open (kokai) 2003-125783); a mutant obtained through substitutionof position 65 of the amino acid sequence of SEQ ID NO: 1 by proline; amutant obtained through substitution of position 101 of the same aminoacid sequence by asparagine; mutants obtained through substitution ofposition 273 of the same amino acid sequence by isoleucine, glycine, orthreonine; mutants obtained through substitution of position 320 of thesame amino acid sequence by phenylalanine, valine, threonine, leucine,isoleucine, or glycine; mutants obtained through substitution ofposition 359 of the same amino acid sequence by serine, leucine, valine,isoleucine, or glutamine, mutants obtained through substitution ofposition 387 of the same amino acid sequence by alanine, lysine,glutamine, glutamic acid, arginine, or histidine (Japanese PatentApplication Laid-Open (kokai) 2004-000122); mutants obtained throughsubstitution of position 163 of the amino acid sequence of SEQ ID NO: 1by histidine, aspartic acid, phenylalanine, lysine, asparagine, serine,isoleucine, leucine, glutamine, threonine or valine; mutants obtainedthrough substitution of position 170 of the same amino acid sequence byvaline or leucine; mutants obtained through substitution of position 171of the same amino acid sequence by alanine, glutamic acid, glycine, orthreonine (Japanese Patent Application Laid-Open (kokai) 2004-057195);and an alkaline protease having an amino acid sequence that exhibits a80% or higher, preferably 87% or more, more preferably 90% or more,still more preferably 95% or more, homology with any of the above listedamino acid sequences.

Homology of amino acid sequences can be preferably determined by theLipman-Pearson method (Science, 227, 1435, 1985).

“Amino acid residues located at positions corresponding to the positions. . . ” can be identified by comparing amino acid sequences of alkalineproteases by means of a known algorithm such as the Lipman-Pearsonmethod, to thereby assign maximum homology to conserved amino acidresidues present in the amino acid sequences. When the amino acidsequences of proteases are aligned by means of such method, regardlessof insertion or deletion occurred in the amino acid sequences, thepositions of the homologous amino acid residues can be determined ineach of the proteases. Conceivably, homologous amino acid residues arelocated at the same positions in the three-dimensional structure ofprotease, whereby analogous effects are obtained in terms of specificfunctions of the intended protease.

As shown in FIG. 1, in which amino acid sequences are aligned by meansof the aforementioned method, the amino acid residue at “(a) position 15of the amino acid sequence of SEQ ID NO: 1” is serine. Through use ofthe method described in the above paragraph, an amino acid residue at aposition corresponding to that position can be identified as, forexample, asparagine at position 15 in case of protease E-1. In thisconnection, the amino acid residue at that position is preferablyhistidine.

The amino acid residue at “(b) position 16 of the amino acid sequence ofSEQ ID NO: 1” is serine. Through use of the above-described method, anamino acid residue at a position corresponding to that position can beidentified as, for example, asparagine at position 16 in case ofprotease E-1. Preferably, the amino acid residue at that position isthreonine or glutamine.

The amino acid residue at “(c) position 166 of the amino acid sequenceof SEQ ID NO: 1” is asparagine. Through use of the above-describedmethod, an amino acid residue at a position corresponding to thatposition can be identified as, for example, asparagine at position 165in case of protease Ya. Preferably, the amino acid residue at thatposition is glycine.

The amino acid residue at “(d) position 167 of the amino acid sequenceof SEQ ID NO: 1” is glycine. Through use of the above-described method,amino acid residue at a position corresponding to that position can beidentified as, for example, serine at position 166 in case of proteaseYa. Preferably, the amino acid residue at that position is valine.

The amino acid residue at “(e) position 187 of the amino acid sequenceof SEQ ID NO: 1” is asparagine. Through use of the above-describedmethod, an amino acid residue at a position corresponding to thatposition can be identified as, for example, asparagine at position 186in case of protease SD-521. Preferably, the amino acid residue at thatposition is serine.

The amino acid residue at “(f) position 346 of the amino acid sequenceof SEQ ID NO: 1” is lysine. Through use of the above-described method,an amino acid residue at a position corresponding to that position canbe identified as, for example, lysine at position 346 in case ofprotease KP9860. Preferably, the amino acid residue at that position isarginine.

The amino acid residue at “(g) position 405 of the amino acid sequenceof SEQ ID NO: 1” is asparagine. Through use of the above-describedmethod, an amino acid residue at a position corresponding to thatposition can be identified as, for example, asparagine at position 405in case of protease KP9860. Preferably, the amino acid residue at thatposition is aspartic acid.

Specific examples of the positions and amino acid residues correspondingto (a) position 15, (b) position 16, (c) position 166, (d) position 167,(e) position 187, (f) position 346, and (g) position 405 of the aminoacid sequence (SEQ ID NO: 1) of protease KP43 and positionscorresponding to these positions are shown below by way of somepreferred examples of the aforementioned “another alkaline protease”(Table 1).

TABLE 1 Protease Position KP43 KP9860 9865 E-1 Ya SD-521 A-1 A-2 (a)Ser15 Ser15 Ser15 Asn15 Asn15 Asn15 Ser15 Asn15 (b) Ser16 Ser16 Ser16Asn16 Asn16 Asn16 Ser16 Asn16 (c) Ser166 Ser166 Ser166 Asn165 Asn165Asn165 Asn166 Gly165 (d) Gly167 Gly167 Gly167 Ser166 Ser166 Ser166Gly167 Ser166 (e) Asn187 Asn187 Asn187 Asn186 Asn186 Asn186 Asn187Asn186 (f) Lys346 Lys346 Lys346 Lys345 Lys345 Lys345 Lys346 Lys345 (g)Asn405 Asn405 Asn405 Asn404 Asn404 Asn404 Asn405 Asn404

Among the positions (a) to (g) of the amino acid residues of thealkaline protease of the present invention, two or more positions of thepositions (a) to (g) may be concurrently selected, so long as the enzymecharacteristics remain unchanged. Preferred examples of two or morepositions being selected concurrently are shown below. Amino acids aredesignated by the three letter codes, and the symbol “+” means anadditional substitution.

Specific examples of two substitutions (i.e., substitution taking placeat two positions) include Ser15His+Ser16Thr, Ser15His+Ser16Gln,Lys346Arg+Asn405Asp, Asn187Ser+Lys346Arg, and Asn166Gly+Gly167Val,wherein Ser15His+Ser16Gln, Lys346Arg+Asn405Asp, and Asn166Gly+Gly167Valare preferred.

Specific examples of three substitutions (i.e., substitution takingplace at three positions) include Ser15His+Ser16Thr+Asn187Ser,Ser15His+Ser16Gln+Asn187Ser, and Asn187Ser+Asn166Gly+Gly167Val, whereinSer15His+Ser16Gln+Asn187Ser and Asn187Ser+Asn166Gly+Gly167Val arepreferred.

So long as the requirements of the present invention are satisfied, fourto seven substitutions may take place, and seven substitutions ofSer15His+Ser16Gln+Asn166Gly+Gly167Val+Asn187Ser+Lys346Arg+Asn405Asp ismore preferred.

When the alkaline protease of the present invention is a mutant, thealkaline protease before undergoing mutagenesis (which may be referredto as a “parent alkaline protease”) is either a “protease having anamino acid sequence of SEQ ID NO: 1” or the aforementioned “anotheralkaline protease.” When the parent alkaline protease is subjected tomutation at a predetermined site thereof, the alkaline protease of thepresent invention can be obtained. For example, when an amino acidresidue at a position selected from the aforementioned positions (a) to(g) of the amino acid sequence of SEQ ID NO: 1 of protease (KP43 or anamino acid residue at a position corresponding to any of the abovepositions in the amino acid sequence of another alkaline protease) isreplaced by another amino acid residue, the alkaline protease of thepresent invention can be obtained.

The alkaline protease of the present invention may be obtained through,for example, the following steps. Briefly, a cloned gene encoding parentalkaline protease (SEQ ID NO: 2; a gene encoding SEQ ID NO: 1, or amature enzyme region, is represented by the sequence starting from the619th codon) is mutated, and by use of the thus-mutated gene anappropriate host bacterium is transformed, followed by culturing of therecombinant host bacterium and collecting the alkaline protease productof the invention from the culture. Cloning of the gene encoding theparent alkaline protease may be carried out through a generally employedgene recombination technique. For example, a method described in PatentPublication WO99/18218 or Patent Publication WO98/56927 may be employed.

Means for carrying out mutagenesis of the gene encoding the parentalkaline protease may be random mutagenesis or site-directed mutagenesiswhich are commonly performed. More specifically, mutagenesis of the genemay be carried out by use of, for example, a Site-Directed MutagenesisSystem Mutan-Super Express Km kit (Takara). Alternatively, by means ofrecombinant PCR (polymerase chain reaction; see “PCR Protocols,”Academic Press, New York, 1990), an arbitrary sequence of the gene canbe replaced by the arbitrary sequence of another gene.

Production of the protease of the present invention by use of thethus-obtained mutant gene may be carried out, for example, by ligatingthe mutated gene to a DNA vector capable of stably amplifying the gene,to thereby transform host bacteria. Alternatively, the mutant gene maybe introduced into chromosomal DNA of a host bacterium capable of stablymaintaining the gene. Examples of the host bacterium which satisfiesthese requirements include bacteria belonging to the genus Bacillus,Escherichia coli, mold, yeast, and actinomycetes. Any of thesemicroorganisms is inoculated into a culture medium containing anassimilable carbon source, nitrogen source, and other essentialnutrients, and culturing is carried out according to a customary method.

From the thus-obtained culture, alkaline protease may be collected andpurified by means of customary methods for collecting and purifyingenzymes. For example, the culture is subjected to centrifugation orfiltration to thereby remove cells, and the enzyme of interest isobtained from the culture supernatant by means of a routine purificationtechnique. The thus-obtained enzyme solution may be employed as is.Alternatively, the enzyme solution may further be subjected topurification, crystallization, powdering, or granulation, any of whichmay be carried out according to a known method.

The thus-produced alkaline protease of the present invention for exampleexhibits oxidant resistance and maintains casein-degrading activity evenin the presence of a fatty acid at a high concentration. The alkalineprotease has a molecular weight of 43,000±2,000 as determined bySDS-PAGE, and is active within the alkaline region. Moreover, thealkaline protease exhibits newly acquired properties; i.e., improvedspecific activity and protein secretion amount compared with those ofthe parent alkaline protease.

Thus, the alkaline protease of the present invention is useful as anenzyme to be incorporated in a variety of detergent compositions.

No particular limitation is imposed on the amount of the protease of thepresent invention to be incorporated into a detergent composition, solong as the alkaline protease exhibits activity. The preferred amount is0.1 to 5,000 PU per kg of detergent composition, more preferably 500 PUor less, in consideration of cost and other factors.

The detergent composition of the present invention may further contain avariety of enzymes in addition to the protease of the present invention.Examples of such additional enzymes include hydrolase, oxidase,reductase, transferase, lyase, isomerase, ligase, and synthetase. Ofthese, preferred enzymes include proteases other than those of thepresent invention, cellulase, keratinase, esterase, cutinase, amylase,lipase, pullulanase, pectinase, mannanase, glucosidase, glucanase,cholesteroloxidase, peroxidase, and laccase, among which the proteases,cellulase, amylase, and lipase are more preferred. Examples of theproteases include commercially available ones that are derived fromBacillus, such as ALCALASE, ESPERASE, SAVINASE, EVERLASE, and KANNASE(all are registered trademarks; Novozymes), PROPERASE and PURAFECT(registered trademarks; Genencor); and KAP (Kao Corp.). Examples ofcellulase include those derived from Humicola such as CELLUZYME andCAREZYME (registered trademarks; Novozymes); and KAC, alkaline cellulaseproduced by Bacillus sp. KSM-S237 disclosed in Japanese PatentApplication Laid-Open (kokai) No. 10-313859, and mutated alkalinecellulase disclosed in Japanese Patent Application Laid-Open (kokai) No.2003-313592 (these are products of Kao Corp.). Examples of amylaseinclude those derived from Bacillus such as TERMAMYL and DURAMYL(registered trademarks; Novozymes), PURASTAR (registered trademark;Genencor), and KAM (Kao Corp.). Examples of lipase are those derivedfrom Thermomyces and include LIPOLASE and LIPOLASE ULTRA (registeredtrademarks; Novozymes).

When a protease species other than the protease of the present inventionis incorporated into a detergent composition together with the proteaseof the present invention, its amount is preferably 0.1 to 500 PU per kgof detergent composition. When cellulase is incorporated in combination,the amount of cellulase is preferably 300 to 3,000,000 KU per kg ofdetergent composition, based on the unit (KU) determined through theenzyme activity determination method described in paragraph [0020] ofJapanese Patent Application Laid-Open (kokai) No. 10-313859.

When amylase is incorporated in combination, its amount is preferably 50to 500,000 IU per kg of detergent composition based on the unit (IU)determined through the amylase activity determination method describedin paragraph [0040] of Japanese Patent Application Laid-Open (kokai) No.11-43690.

Moreover, when lipase is incorporated in combination, its amount ispreferably 10,000 to 1,000,000 LU per kg of detergent composition basedon the unit (LU) determined through the lipase activity determinationmethod described in Example 1 of Japanese Kohyo (PCT) Patent PublicationNo. 8-500013.

Known detergent components may be incorporated into the detergentcomposition of the present invention. Examples of such known detergentcomponents include the following substances.

(1) Surfactant

Generally, a surfactant is incorporated into the detergent compositionin an amount of 0.5 to 60 mass %. In particular, the amount ofsurfactant is preferably 10 to 45 mass % for preparing a powderydetergent composition, and 20 to 50 mass % for preparing a liquiddetergent composition. When the detergent composition of the presentinvention serves as a bleach composition or a detergent composition foran automated dishwasher, a surfactant is typically incorporated in anamount of 1 to 10 mass %, preferably 1 to 5 mass %.

Examples of the surfactant employed in the detergent composition of thepresent invention include an anionic surfactant, a nonionic surfactant,an amphoteric surfactant, a cationic surfactant, and a combinationthereof. Of these, an anionic surfactant and a nonionic surfactant arepreferred.

Examples of a preferred anionic surfactant include a sulfate ester saltof C10-C18 alcohol, a sulfate ester salt of an alkoxylated product ofC8-C20 alcohol, an alkylbenzenesulfonate salt, a paraffinsulfonate salt,an α-olefinsulfonate salt, an α-sulfo fatty acid salt, and an α-sulfofatty acid alkyl ester salt or a fatty acid salt. In the presentinvention, a linear C10-C14 (preferably C12-C14) alkylbenzenesulfonicacid salt is more preferred. The counter ion is preferably an alkalimetal or an amine, and sodium and/or potassium, monoethanol amine, ordiethanol amine is more preferred.

Examples of a preferred nonionic surfactant include a polyoxyalkylenealkyl (C8-C20) ether, an alkylpolyglycoside, a polyoxyalkylene alkyl(C8-C20) phenyl ether, a polyoxyalkylene sorbitan fatty acid (C8-C22)ester, a polyoxyalkylene glycol fatty acid (C8-C22) ester, and apolyoxyethylene polyoxypropylene block polymer. A more preferrednonionic surfactant is a polyoxyalkylene alkyl ether [having an HLBvalue (as calculated through the Griffin method) of 10.5 to 15.0,preferably 11.0 to 14.5] which is obtained by adding 4 to 20 moles ofalkyleneoxide (e.g., ethyleneoxide and propyleneoxide) to a C10-C₁₋₈alcohol.

(2) Divalent Metal Ion Scavenger

A divalent metal ion scavenger is preferably incorporated into thecomposition in an amount of 0.01 to 50 mass %, preferably 5 to 40 mass%. Examples of the divalent metal ion scavenger to be employed in thedetergent composition of the present invention include a condensedphosphate such as a tripolyphosphate, pyrophosphate, or orthophosphate;an aluminosilicate such as zeolite; a synthesized layered crystallinesilicate; a nitrilotriacetate; an ethyenediamineteraacetate; a citrate;an isocitrate; and a polyacetalcarboxylate. Of these, a crystallinealuminosilicate (synthesized zeolite) is more preferred. Among A-type,X-type, and P-type zeolites, A-type zeolite is particularly preferred.The synthesized zeolite preferably has an average primary particle sizeof 0.1 to 10 μm, more preferably 0.1 to 5 μm.

(3) Alkaline Agent

An alkaline agent is preferably incorporated into the composition in anamount of 0.01 to 80 mass %, preferably 1 to 40 mass %. Examples of thealkaline agent which may be incorporated into the detergent in powderform include an alkali metal carbonate such as sodium carbonate(collectively referred to as dense ash or light ash) and an amorphousalkali metal silicate such as JIS No. 1, No. 2, or No. 3. Theseinorganic alkaline agents are effective for the formation of theskeleton of particles during drying of the detergent, contributing toproduction of a detergent of relatively hard particles with excellentflowability. Examples of alkaline agents other than the above-describedsubstances include sodium sesquicarbonate and sodium hydrogencarbonate.A phosphate such as tripolyphosphate also acts as an alkaline agent.Examples of alkaline agents to be employed in a detergent in liquid forminclude, in addition to the above-described alkaline agents, sodiumhydroxide and mono-, di-, or tri-ethanol amine, which can also beemployed as a counter ion of a surfactant.

(4) Anti-Redeposition Agent

An anti-redeposition agent is preferably incorporated into thecomposition in an amount of 0.001 to 10 mass %, preferably 1 to 5 mass%. Examples of the anti-redeposition agent to be employed in thedetergent composition of the present invention include a polyethyleneglycol, a carboxylic polymer, a polyvinyl alcohol, and a polyvinylpyrrolidone. Of these, the carboxylic polymer exerts not only ananti-redeposition effect, but also the effect of scavenging metal ionsand the effect of releasing solid soil particles from the clothing intothe washing liquid. The carboxylic polymer is a homopolymer or acopolymer of, for example, acrylic acid, methacrylic acid, or itaconicacid. Examples of preferred copolymers include a copolymerized productof any of the above monomers and maleic acid. The copolymer preferablyhas a molecular weight of some thousands to 100,000. In addition to theabove carboxylic polymers, a polymer such as poly(glycidyl acid salt), acellulose derivative such as carboxymethyl cellulose, and anaminocarboxylic polymer such as poly(aspartic acid) are also preferred,since these substances function as a metal ion scavenger, a dispersingagent, and an anti-redeposition agent.

(5) Bleaching Agent

A bleaching agent such as hydrogen peroxide or a percarbonate ispreferably incorporated into the composition, preferably in an amount of1 to 10 mass %. When such a bleaching agent is employed,teraacetylethylenediamine (TAED) or a bleaching activator described in,for example, Japanese Patent Application Laid-Open (kokai) No. 6-316700may be incorporated into the composition in an amount of 0.01 to 10 mass%.

(6) Fluorescent Agent

Examples of a fluorescent agent which may be incorporated into thedetergent composition of the present invention include a biphenylfluorescent agent (e.g., Tinopal CBS-X) and a stilbene fluorescent agent(e.g., DM-type fluorescent agent). The fluorescent agent is preferablyincorporated in an amount of 0.001 to 2%.

(7) Other Components

The detergent composition of the present invention may contain abuilder, a softener, a reducing agent (e.g., sulfite), a deformer (e.g.,silicone), a perfume, or other additives, which are known in the fieldof laundry detergents.

The detergent composition of the present invention can be producedthrough a routine method by using, in combination, the protease productof the present invention obtained through the above-described method andknown detergent components as listed above. The form of the detergentmay be determined in accordance with its use, and examples of the forminclude liquid, powder, granules, paste, and solid.

The thus-obtained detergent composition of the present invention can beused as, among others, a laundry detergent, a bleaching agent, adetergent for hard surfaces, a drainpipe detergent, a denture detergent,or a germicidal detergent for medical instruments.

EXAMPLES

The following examples further describe and demonstrate embodiments ofthe present invention. The examples are given solely for the purpose ofillustration and are not to be construed as limitations of the presentinvention.

Example 1

A region of about 2.0 kb up to the stop codon of an alkaline proteasestructural gene derived from Bacillus sp. KSM-KP43 was subjected toerror prone PCR by use of a Takara Taq (product of Takara), which lackserror repair ability, and by use of adequate amounts of manganesesulfate and dimethylsulfoxide, whereby random mutagenesis wasintroduced. PCR was carried out using primer 1 (SEQ ID NO: 4) and primer2 (SEQ ID NO: 4) capable of amplifying the above mentioned DNA fragmentof about 2.0 kb, wherein primer 1 was a sense primer with BamHI linkerat the 5′ end, and primer 2 was an antisense primer with linker at the5′ end. In PCR, the template DNA was denatured at 94° C. for one minute,followed by 30 cycles of treatment, each cycle consisting of 94° C.×oneminute, 55° C.×one minute, and 72° C.×two minutes. The amplified DNAfragments were purified by use of a DNA Product Purification kit(Roche), and the terminal restriction endonuclease linkers were cleavedwith BamHI and XbaI. The amplified DNA was mixed with plasmid pHA64which had undergone treatment with BamHI and XbaI (see Japanese PatentApplication Laid-Open (kokai) 2000-287687; BamHI- and XbaI-cleaved sitesare contained in a downstream region of promoter 64), and subsequently,ligation reaction was carried out with Ligation High (product ofToyobo). Through ethanol precipitation, plasmid was recovered from theligase reaction mixture.Bacillus sp. KSM-9865 (FERM P-18566) serving asthe host bacterium was transformed.

The KSM-9865 cells which had undergone the transformation step weregrown on a skim milk-containing alkaline agar medium [skim milk (Difco)(1% (w/v)), bactotryptone (Difco) (1%), yeast extract (Difco) (0.5%),sodium chloride (0.5%), agar (1.5%), sodium carbonate (0.05%), andtetracycline (15 ppm)]. Whether or not a mutated protease gene had beenintroduced to the KSM-9865 cells was determined on the basis of haloformation. The resultant transformants were inoculated into a seedculture medium (5 mL) [6.0% (w/v) polypeptone S, 0.05% of yeast extract,1.0% of maltose, 0.02% of magnesium sulfate heptahydrate, 0.1% ofpotassium dihydrogenphosphate, 0.25% of sodium carbonate, and 30 ppm oftetracycline], followed by shaking the culture for 16 hours at 30° C.The seed culture broth (1% (v/v)) was inoculated into a main culturemedium (30 mL) [8% polypeptone S, 0.3% of yeast extract, 10% of maltose,0.04% of magnesium sulfate heptahydrate, 0.2% of potassiumdihydrogenphosphate, 1.5% of sodium carbonate anhydrate, and 30 ppmtetracycline], followed by shaking the culture for three days at 30° C.

The resultant culture was subjected to centrifugation, and the proteaseactivity of the culture supernatant was measured. Protease activity wasmeasured by means of a method employing casein as a substrate, and theprotein amount was measured by use of a protein assay kit (Wako PureChemical Industries, Ltd.). Through comparison between the obtainedmeasurement and a measurement obtained from a culture supernatant of aculture (culturing was performed under the same culture conditions asabove) of a transformant harboring a wild type enzyme gene, mutantprotease genes showing improved protease activity were selected.

From the selected transformants, plasmid was recovered by use of a HighPure Plasmid Isolation kit (Roche) and subjected to nucleotidesequencing. By use of plasmid DNA (300 ng) as a template, PCR wascarried out in a 20 μL reaction system employing a Big Dye DNAsequencing kit (Applied Biosystems). For analysis, a DNA Sequencer(model: 377, Applied Biosystems) was used. As a result, improvedprotease productivity was found in the following mutants: an enzyme inwhich serine at position 15 was replaced by histidine, an enzyme inwhich serine at position 16 was replaced by threonine, a double mutationin which serine at position 15 and serine at position 16 were replacedby histidine and glutamine, respectively, an enzyme in which asparagineat position 166 was replaced by glycine, an enzyme in which glycine atposition 167 was replaced by valine, a double mutation in whichasparagine at position 166 and glycine at position 167 were replaced byglycine and valine, respectively, an enzyme in which asparagine atposition 187 was replaced by serine, an enzyme in which lysine atposition 346 was replaced by arginine, an enzyme in which asparagine atposition 405 was replaced by aspartic acid, and a double mutation inwhich lysine at position 346 and asparagine at position 405 werereplaced by arginine and aspartic acid, respectively. Of these mutants,those exhibiting particularly improved productivity are a doublemutation in which serine at position 15 and serine at position 16 werereplaced by histidine and glutamine, respectively, a double mutation inwhich asparagine at position 166 and glycine at position 167 werereplaced by glycine and valine, respectively, a double mutation in whichlysine at position 346 and asparagine at position 405 were replaced byarginine and aspartic acid, respectively, and an enzyme in whichasparagine at position 187 was replaced by serine.

A portion of the culture was diluted, and the diluted portion wasapplied to DEAE-TOYOPEARL (an anion-exchange gell for ion-exchangechromatography manufactured by Tosoh) equilibrated with 10-mM Tris-HClbuffer containing 2-mM CaCl₂ (pH of the buffer system: 7.5). Anon-adsorbed fraction was recovered, whereby substantially homogeneousprotease was obtained. Protein amount and casein degradation activitywere measured for each purified enzyme. The measurements show thatimprovement in productivity was attained by the mutations introduced,which led to an improved amount of secreted protein (102 to 108%) or animproved specific activity (104 to 121%), on the basis of the secretionamount or the specific activity of a wild type enzyme being taken as100% (Table 2).

Next, in an attempt to combine the above-described individual mutationsites, recombinant PCR was performed through use of primers 1 to 8 (SEQID NOs: 4 to 11) and Pyrobest (Takara), whereby mutants each bearingcombinatorial mutation sites were created. Briefly, using as a templatea wild type gene or a mutant gene, a DNA fragment having a size of about700 bp (including the 15th and 16th positions) from the translationinitiation site of the alkaline protease structural gene was amplifiedwith primer 1 (SEQ ID NO: 4) and primer 3 (SEQ ID NO: 6) forcombinatorial mutation, to thereby create a mutant. Similarly, a DNAfragment having a size of about 500 bp including positions 166 and 167was amplified with primer 4 (SEQ ID NO: 7) and primer 5 (SEQ ID NO: 7);a DNA fragment having a size of 400 bp including position 187 wasamplified with primer 6 (SEQ ID NO: 9) and primer 7 (SEQ ID NO: 10); anda DNA fragment having a size of about 500 bp (including positions 346and 405) up to the termination codon of the alkaline protease structuralgene was amplified with primer 2 (SEQ ID NO: 5) and primer 8 (SEQ ID NO:11). Respective mutants were incubated and then assessed for proteaseproductivity. Improvement in productivity was confirmed onS15H/S16Q/K346R/N405D, S15H/S16Q/N187S/K346R/N405D, andS15H/S16Q/N166G/G167V/N187S/K346R/N405D. The results show thatimprovement in productivity was attained by the enhanced amount ofsecreted protein (107 to 112%) or an improved specific activity (103 to115%) in respective mutants, on the basis of the secretion amount or thespecific activity of a wild type enzyme being taken as 100% (Table 2).

TABLE 2 Relative Relative protein specific secretion activity (%) (%)Wild type 100 100 S15H + S16T 102 101 S15H + S16Q 108 104 N166G + G167V100 121 N187S 101 108 K346R + N405D 102 107 S15H + S16Q + K346R + N405D112 103 S15H + S16Q + N187S + K346R + N405D 107 113 S15H + S16Q +N166G + G167V + 111 115 N187S + K346R + N405D

The above-listed example alkaline protease mutants of the presentinvention were found to attain either enhanced protein secretion amountwith respect to casein or improved specific activity, or both. Exceptfor these new characteristics, they were found to exhibit thecharacteristics of the parental alkaline protease; i.e., they exhibitoxidant resistance, maintain casein-degrading activity even in thepresence of a fatty acid of high concentration, have a molecular weightof 43,000±2,000 as determined by SDS-PAGE, and are active within thealkaline region.

Referential Example

Protease Assay (Casein Method)

A 50 mM borate buffer (pH 10.5) (1 mL) containing casein (Hammersteinmethod: Merck, 1% (w/v)) was maintained at 30° C. for five minutes, andsubsequently an enzyme solution (0.1 mL) was added to the buffer, tothereby allow reaction to proceed for 15 minutes. A reaction stoppingsolution (0.11M trichloroacetic acid/0.22M sodium acetate/0.33M aceticacid) (2.0 mL) was added to the resultant reaction mixture, and themixture was allowed to stand at room temperature for 30 minutes.Thereafter, the filtrate was collected through filtration by use of aWhatman No. 1 filter, and the degradation product was quantified bymeans of the method described by Lowry, et al. Specifically, an alkalinecopper solution (1% Rochelle salt: 1% of copper sulfate pentahydrate: 2%of sodium carbonate/0.1N sodium hydroxide solution=1:1:100) (2.5 mL) wasadded to the filtrate (0.5 mL), and the resultant mixture was allowed tostand at 30° C. for 10 minutes. Subsequently, to the mixture was added aphenol reagent [obtained by diluting a commercial phenol reagent (KantoKagaku) two-fold with deionized water] (0.25 mL), and the resultantmixture was thoroughly stirred and left to stand at 30° C. for 30minutes. Thereafter, the absorbance of the mixture was measured at 660nm. One unit of protease activity (1 PU) was defined as the amount ofenzyme required for producing acid-soluble protein equivalent to 1 mmolof tyrosine per minute under the above reaction conditions.

Example 2

(1) Preparation of Detergent

Water (465 kg) was added to a mixing bath (1 m³) equipped with astirring paddle. After the temperature of the water reached 55° C., a40% (w/v) sodium polyacrylate aqueous solution (135 kg) was added to thewater. The resultant mixture was stirred for 15 minutes, and then sodiumcarbonate (120 kg), sodium sulfate (60 kg), sodium sulfite (9 kg), and afluorescent dye (3 kg) were added to the mixture. The resultant mixturewas further stirred for 15 minutes, and zeolite (300 kg) was added tothe mixture, followed by stirring for 30 minutes, to thereby yield ahomogenous slurry (the water content of the slurry: 50 mass %). Theslurry was sprayed through pressure spray nozzles provided in thevicinity of the top of a spray-drying tower, to thereby yield a granularbase (a high-temperature gas was fed at 225° C. through a lower part ofthe spray-drying tower, and discharged at 105° C. from the top of thetower).

Subsequently, the thus-obtained granular base (100 parts by mass) wasfed to a Lodige mixer (product of Matsuzaka Giken Co., Ltd., capacity:20 L, equipped with a jacket). While the granular base was stirred bymeans of rotation of the main shaft (150 rpm), a mixture of a nonionicsurfactant (20 parts by mass), sodium linear alkyl (C10-C13)benzenesulfonate (22 parts by mass), a fatty acid (C14-C18) sodium salt(4 parts by mass), polyethylene glycol (2 parts by mass), and water (4parts by mass) were added to the mixer over three minutes. Thereafter,the resultant mixture was stirred for five minutes. Furthermore,crystalline sodium silicate (20 parts by mass) and zeolite (10 parts bymass) were added to the mixer for surface coating, to thereby yield adetergent base.

The detergent base (99 mass %) was mixed with example protease granulesof the present invention (0.5 mass %) and a perfume (0.5 mass %), tothereby produce an end product, granular detergent A.

(2) Raw Materials Employed

Nonionic surfactant: Emulgen 108KM (average mole number of ethyleneoxide added: 8.5, product of Kao Corporation)

Aqueous solution of sodium polyacrylate: average molecular weight:10,000 (produced by use of the method described in Examples of JapanesePatent Publication (kokoku) No. 2-24283)

Sodium carbonate: Dense ash (product of Central Glass Co., Ltd.)

Zeolite: Zeolite 4A (average particle size: 3.5 μm, product of TosohCorporation)

Polyethylene glycol: K-PEG6000 (average molecular weight: 8,500, productof Kao Corporation)

Crystalline sodium silicate: Powder SKS-6 (product of Hoechst Tokuyama)

Example protease granules of the present invention: granules preparedfrom each of the purified samples of the example alkaline proteases ofthe present invention shown in Table 2 by use of the method described inExample 1 of Japanese Patent Application Laid-Open (kokai) No. 62-257990(6 PU/g)

Fluorescent dye: TINOPAL CBS-X (a DSBP-type Fluorescent Whitening Agentwhich is product of Ciba-Geigy Corp.)

Example 3

(1) Preparation of Detergent

The slurry (solid content: 50 mass %) was spray-dried with 250° C. hotair, to thereby yield a granular base containing sodium polyacrylate(mass average molecular weight: 10,000) (7 mass %), sodium carbonate (26mass %), sodium sulfate (20 mass %), sodium chloride (6 mass %), thefluorescent dye (0.5 mass %), zeolite (40 mass %), and water (0.5 mass%).

Subsequently, the thus-obtained granular base (100 parts by mass) wasfed to a Lodige mixer (product of Matsuzaka Giken Co., Ltd., capacity:20 L, equipped with a jacket). While the granular base was stirred bymeans of rotation of the main shaft (150 rpm), a mixture of a nonionicsurfactant (20 parts by mass), sodium linear alkyl (C10-C13)benzenesulfonate (22 parts by mass), a fatty acid (C14-C18) sodium salt(4 parts by mass), polyethylene glycol (2 parts by mass), and water (4parts by mass) were added to the mixer over three minutes. Thereafter,the resultant mixture was stirred for five minutes. Furthermore,crystalline sodium silicate (20 parts by mass) and zeolite (10 parts bymass) were added to the mixer for surface coating, to thereby yield adetergent base.

The detergent base (95 mass %) was mixed with bleaching agent granules(2.8 mass %), bleaching activator granules (1.2 mass %), exampleprotease granules of the present invention (0.5 mass %), and a perfume(0.5 mass %), to thereby produce an end product, granular detergent B.

(2) Raw Materials Employed

Nonionic surfactant: Emulgen 108KM (average mole number of ethyleneoxide added: 8.5, product of Kao Corporation)

Aqueous solution of sodium polyacrylate: average molecular weight:10,000 (produced by use of the method described in Examples of JapanesePatent Publication (kokoku) No. 2-24283)

Sodium carbonate: Dense ash (product of Central Glass Co., Ltd.)

Zeolite: Zeolite 4A (average particle size: 3.5 μm, product of TosohCorporation)

Polyethylene glycol: K-PEG6000 (average molecular weight: 8,500, productof Kao Corporation)

Crystalline sodium silicate: SKS-6 (product of Hoechst Tokuyama)

Example protease granules of the present invention: granules preparedfrom each of the purified samples of the example alkaline proteases ofthe present invention shown in Table 2 by use of the method described inExample 1 of Japanese Patent Application Laid-Open (kokai) No. 62-257990(6 PU/g)

Fluorescent dye: TINOPAL CBS-X (a DSBP-type Fluorescent Whitening Agentwhich is product of Ciba-Geigy Corp.)

Example 4

Liquid detergent compositions (detergents C and D) shown in Table 3 wereprepared.

TABLE 3 Detergent C Detergent D Components (mass %) (mass %) Nonionicsurfactant¹⁾ 25.0  — Nonionic surfactant²⁾ 5.0 — Nonionic surfactant³⁾10.0  — Nonionic surfactant⁴⁾ — 9.0 Nonionic surfactant⁵⁾ — 9.0 Nonionicsurfactant⁶⁾ — 2.5 Anionic surfactant⁷⁾ 1.0 — Silicone⁸⁾ — 0.8Carboxylic acid-based polymer⁹⁾ 2.0 — Polymer¹⁰⁾ — 0.8 Citric acid 0.2 —Calcium chloride  0.05 — Monoethanolamine 4.0 — Triethylene glycolphenyl ether 3.0 — Propylene glycol 3.0 — Ethanol 2.0 2.0 Sodium sulfite0.2 — Example protease of the present 0.5 1.0 invention¹¹⁾ Perfume 0.50.5 Water Balance Balance Total 100    100    Concentration upon use 20g/30 L 40 g/30 L pH of detergent solution 10.5  7.3 ¹⁾Polyoxyethylene(average mole number added: 7) alkyl ether having an alkyl group,derived from a C12-C14 secondary alcohol (SOFTANOL 70, product of NipponShokubai Kagaku Kogyo) ²⁾Polyoxyethylene (average mole number added: 12)alkyl ether having an alkyl group, derived from a C12-C14 secondaryalcohol (SOFTANOL 120, product of Nippon Shokubai Kagaku Kogyo) ³⁾Aproduct obtained by sequentially adding EO (average mole number: 5), PO(average mole number: 2), and EO (average mole number: 3) to a C10-C14linear primary alcohol ⁴⁾Polyoxyethylene lauryl ether (average molenumber of EO added: 8) ⁵⁾Polyoxyethylene lauryl ether (average molenumber of EO added: 11.5) ⁶⁾Narrow range polyoxyethylene alkyl(sec-C₁₂/C₁₃) ether ⁷⁾Sodium linear alkyl (C10-C14) benzenesulfonate⁸⁾Amide/ether-modified silicone polymer (BY16-906, product of DowCorning Toray Silicone Co., Ltd.) ⁹⁾A phenoxypolyethylene glycol -acrylic acid - maleic acid copolymer synthesized by use of the methoddescribed in lines 6 through 13 of page 11 of Japanese PatentApplication Laid-Open (kokai) No. 10-60476 (mass average molecularweight: 10,000, solid content: 51.2%) ¹⁰⁾A sodium salt of apentene/maleic acid (ratio by mol: 50/50) copolymer (mass averagemolecular weight: 7,000) ¹¹⁾A purified sample of each of the examplealkaline proteases of the present invention shown in Table 2 (15 PU/mL)

Example 5

While sodium percarbonate and sodium carbonate (dense ash) of thecomponents shown in Table 4 below were mixed under stirring, a 40%aqueous solution of sodium polyacrylate and sodium linear alkylbenzenesulfonate, a nonionic surfactant, or sodiumlauroyloxybenzenesulfonate were added to the mixture. Subsequently, tothe resultant mixture were added example protease granules of thepresent invention prepared by use of the method described in Example 1of Japanese Patent Application Laid-Open (kokai) No. 62-257990, and theresultant mixture was stirred until a uniform mixture was attained,whereby a bleaching agent was obtained.

TABLE 4 Bleaching Bleaching agent E agent F Components (mass %) (mass %)Sodium percarbonate¹⁾ 72.0 72.0 Sodium carbonate (dense ash) 20.0 20.0Anionic surfactant²⁾ 2.0 — Nonionic surfactant³⁾ — 2.0 Sodiumpolyacrylate⁴⁾ 1.0 1.0 Sodium lauroyloxybenzenesulfonate 4.0 4.0 Exampleprotease of the present 1.0 1.0 invention⁵⁾ ¹⁾Particle size: 500 to 700μm ²⁾Sodium linear alkyl (C12-C14) benzenesulfonate ³⁾Polyoxyethylenealkyl ether (number of carbon atoms of the alkyl group: 12 to 14,average mole number of EO added: 12) ⁴⁾Average molecular weight: 8,000⁵⁾Granules (6 PU/g) prepared from each of the purified samples of theexample alkaline proteases of the present invention shown in Table 2 byuse of the method described in Example 1 of Japanese Patent ApplicationLaid-Open (kokai) No. 62-257990

Example 6

Detergent compositions for an automatic dishwasher (detergents G and H)shown in Table 5 below were prepared.

TABLE 5 Detergent G Detergent H Components (mass %) (mass %) PluronicL-61¹⁾ — 4.0 Softanol EP-7085²⁾ 4.0 — Trisodium citrate — 30.0 Sodiumtripolyphosphate 30.0 — Sodium percarbonate 20.0 20.0 Sodium carbonate20.0 20.0 Amorphous silicate³⁾ 10.0 10.0 AA-MA⁴⁾ 4.0 4.0 Sodium sulfate10.0 10.0 α-Amylase⁵⁾ 1.0 1.0 Example protease of the present 1.0 1.0invention⁶⁾ ¹⁾Polyoxyethylene - polyoxypropylene copolymer (averagemolecular weight: 2,000) ²⁾A product obtained by adding to a C12-C14sec-alcohol ethylene oxide (7 mol) and propylene oxide (8.5 mol) ³⁾JISNo. 2 sodium silicate ⁴⁾An acrylic acid - maleic acid copolymer⁵⁾Duramyl 60T (registered trademark; product of Novozymes) ⁶⁾Granules (6PU/g) prepared from each of the purified samples of the example alkalineproteases of the present invention shown in Table 2 by use of the methoddescribed in Example 1 of Japanese Patent Application Laid-Open (kokai)No. 62-257990

Example 7

A detergent composition for hard surfaces (detergent J) was preparedfrom components shown in Table 6 below.

TABLE 6 Components Detergent J (mass %) Anionic surfactant¹⁾ 15.0Nonionic surfactant²⁾ 5.0 Nonionic surfactant³⁾ 5.0 Amphotericsurfactant⁴⁾ 7.5 Amphoteric surfactant⁵⁾ 4.0 Citric acid 1.0Polypropylene glycol⁶⁾ 2.0 Ethanol 5.0 Example protease of the present1.0 invention⁷⁾ Perfume, water, etc./pH modifier 54.5 Total 100.0¹⁾Sodium polyoxyethylene (EOP = 4) alkyl (C12) ether sulfate²⁾Polyoxyethylene (EOP = 8) alkyl (C12) ether ³⁾Alkyl (C12)polyglucoside (condensation degree: 1.3) ⁴⁾Mono long-chain tertiaryalkyl (C12) dimethylamine oxide ⁵⁾Alkyl (C12) hydroxydimethylsulfobetaine ⁶⁾Molecular weight: 10,000 ⁷⁾Each of the purified samplesof the example alkaline protease of the present invention shown in Table2 (15 PU/mL)

Example 8

Granular detergents shown in Table 7 below were prepared by use of theaforementioned detergent A (see Example 2).

TABLE 7 Components (mass %) Detergent K Detergent L Detergent MDetergent N Detergent base 98.4 98.3 98.5 97.2 of Example 2 Perfume 0.50.5 0.5 0.5 Example 0.5 0.5 0.5 0.5 protease of the present invention¹⁾Conventional 0.6 0.6 protease²⁾ Cellulase³⁾ 0.7 0.7 Lipase⁴⁾ 0.5 0.5¹⁾Granules (6 PU/g) prepared from each of the purified samples of theexample alkaline proteases of the present invention shown in Table 2 byuse of the method described in Example 1 of Japanese Patent ApplicationLaid-Open (kokai) No. 62-257990 ²⁾Protease K-16 described in JapanesePatent Application Laid-Open (kokai) No. 5-25492, the activity thereofhaving been regulated to 5 PU/g by use of the method described inExample 1 of Japanese Patent Application Laid-Open (kokai) No. 62-257990³⁾KAC-500 (registered trademark; a cellulase derived form Humicola whichis a product of Kao Corporation) ⁴⁾LIPOLASE 100T (registered trademark;a lipase derived from Thermomyces which is a product of Novozymes)

The present invention enables production of an alkaline protease forexample which exhibits activity even in the presence of a fatty acid ofhigh concentration and excellent detergency against complex soilscontaining not only proteins but also, for example, sebum. Such alkalineprotease is secreted efficiently into culture medium, and has highproductivity.

1. An isolated alkaline protease consisting of an amino acid sequence ofSEQ ID NO:1, except wherein one or more amino acid residues selectedfrom those located at (a) position 15, (b) position 16, (c) position166, (d) position 167, (e) position 187, (f) position 346, and (g)position 405 of the amino acid sequence of SEQ ID NO:1 are replaced withthe following amino acid residues, respectively: (a) histidine, (b)threonine or glutamine, (c) glycine, (d) valine, (e) serine, (f)arginine, and (g) aspartic acid, wherein said alkaline protease hasalkaline protease activity.
 2. An isolated alkaline protease consistingof an amino acid sequence that is at least 95% homologous to the aminoacid sequence of SEQ ID NO: 1, wherein one or more amino acid residuesselected from those located at (a) position 15, (b) position 16, (c)position 166, (d) position 167, (e) position 187, (f) position 346, and(g) position 405 of the amino acid sequence of SEQ ID NO: 1, or atpositions corresponding to these positions, are the following amino acidresidues or are replaced with the following amino acid residues,respectively: (a) histidine, (b) threonine or glutamine, (c) glycine,(d) valine, (e) serine, (f) arginine, and (g) aspartic acid, whereinsaid alkaline protease has alkaline protease activity.